Center for Veterinary Biologics SAM 504.07

Testing Protocol Page 1 of 7 

United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 504

Supplemental Assay Method for the Manual Determination 

of Protein Content of Veterinary Biologics (Biuret)

Date: June 17, 2022 

Number: SAM 504.07 

Supersedes: SAM 504.06, February 8, 2016 

Contact: Email: Methodsrequest.notification@usda.gov 

 Phone: Center for Veterinary Biologics, 515-337-6100 

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844 

Ames, IA 50010 

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by 

USDA and does not imply its approval to the exclusion of other products that may be suitable.

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Testing Protocol Page 2 of 7 

Supplemental Assay Method for the Manual Determination of Protein Content

in Veterinary Biologics (Biuret)

Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training 

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the sample 

4. Performance of the Test

4.1 Standard curve

4.2 Test method

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions

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Testing Protocol Page 3 of 7 

Supplemental Assay Method for the Manual Determination of Protein Content

in Veterinary Biologics (Biuret)

1. Introduction

This Supplemental Assay Method (SAM) describes the classical biuret procedure for the indirect 

determination of the protein content of various veterinary biologics products (i.e., serum, 

antiserum and antitoxins). Protein content is often utilized in the evaluation of such products. 

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below. 

2.1.1 Spectrophotometer or colorimeter (1-cm or greater path length) with 

appropriate accessories, i.e., cuvettes

2.1.2 Common laboratory apparatus and glassware – pipettes, pipettors with 

tips, screw cap tubes, Class A volumetric flasks, linear graph paper 

2.1.3 Computer – with linear regression program (optional) 

2.2 Reagents/supplies 

Equivalent reagents or supplies may be substituted for any brand name listed below. 

2.2.1 Potassium phosphate, monobasic 

2.2.2 Sodium phosphate dibasic, anhydrous 

2.2.3 Sodium chloride 

2.2.4 Sodium phosphate, dibasic

2.2.5 Copper sulfate, pentahydrate 

 

2.2.6 Potassium sodium tartrate

2.2.7 Sodium hydroxide 

2.2.8 Potassium iodide 

2.2.9 Water – deionized, demineralized, reverse osmosis or equivalent in purity 

to water filtered through a Milli-Q purification system 

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Supplemental Assay Method for the Manual Determination of Protein Content

in Veterinary Biologics (Biuret)

2.2.10 Standard protein solution – Crystalline bovine albumin containing a 

known amount of protein 

2.2.11 Bovine serum reference – Normal bovine serum 

3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel must have experience or training in this protocol. This includes 

working knowledge of the use of general laboratory equipment, glassware and chemical 

safety; and specific training in the operation of the laboratory equipment and reagents 

listed in Section 2. 

Analysts performing this procedure should first conduct at least 2 trial runs using controls 

and standards and obtain results within acceptable limits. 

3.2 Preparation of equipment/instrumentation

All equipment must be operated according to manufacturers’ recommendations and 

monitored in compliance with applicable standard operating procedures. 

Turn on spectrophotometer and allow instrument to "warm up" for at least 30 minutes.

3.3 Preparation of reagents/control procedures

3.3.1 Phosphate buffered saline (PBS), 0.01M, pH 7.2 - 7.4: Dissolve 0.34 g 

potassium phosphate, monobasic; 1.10 g sodium phosphate dibasic, anhydrous; 

8.50 g sodium chloride; and 0.15 g sodium phosphate, dibasic in 400-500 mL 

water in a 1-L volumetric flask. QS to volume. Transfer to a 1-L glass bottle and 

sterilize by autoclave. Store at 4°± 5°C, stable for 6 months. (An acceptable 

alternative would be to use a prepared product, such as National Centers for 

Animal Health (NCAH) Media #30033.)

3.3.2 Biuret Reagent: Dissolve 1.50 ± 0.01 g copper sulfate, pentahydrate in 

400 mL water in a 1-L volumetric flask. Add 6.00 ± 0.01 g potassium sodium 

tartrate and mix until dissolved. In a separate container dissolve 30.0 ± 0.01 g 

sodium hydroxide in 300 mL water, and then add to previous solution. Add 1.00 ± 

0.01 g potassium iodide to and QS to 1 L. Store at room temperature, stable for 6 

months. (An acceptable alternative would be to use a prepared product, such as 

NCAH Media #10307.) 

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Supplemental Assay Method for the Manual Determination of Protein Content

in Veterinary Biologics (Biuret)

Critical control point: Biuret reagent should be replaced when crystals or 

other precipitates appear in the solution.

3.3.3 Bovine serum – not sterilized: Donor bovine serum, mix thoroughly, 

dispense 3-mL into 5-mL serum vials. Store at -20°± 10°C, stable for 1 year. (An 

acceptable alternative would be to use a prepared product, such as NCAH Media 

#40032.) 

3.3.4 Standard solutions: Dilute Bovine Albumin with PBS to contain 10 

mg/mL protein. Use 10 mg/mL protein solution as stock for curve. Dilute as listed 

below to establish a working standard curve. 

CONC.(mg/mL) mL STOCK mL PBS

10 1.0 0 

 8 0.8 0.2 

 6 0.6 0.4 

 4 0.4 0.6 

 2 0.2 0.8 

 1 0.1 0.9 

3.4 Preparation of the sample 

Samples are normally sera, antisera or antitoxins, or serum fractions. Occasionally, biuret 

tests are run on other solutions, such as antigens. Follow sample receipt procedures as 

described by standard operating procedures 

4. Performance of the Test

4.1 Standard curve

Run duplicate tubes of each standard solution (Section 3.3.4) using the test method 

described in Section 4.2 to establish a standard curve. Plot average optical density (OD) 

for each point on graph paper (concentration vs. OD) or enter data into computer program 

to plot curve and calculate test results. If OD values for any point differ more than 0.05, 

disregard that point. If more than one point has unacceptable OD variations, rerun 

standard curve. 

Critical control point: A standard curve is accurate for that lot of Biuret reagent. A 

comparison run or a new curve must be run when a new lot is used.

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Supplemental Assay Method for the Manual Determination of Protein Content

in Veterinary Biologics (Biuret)

4.2 Test method 

4.2.1 Dilute sample and bovine serum control 1:10 or 1:20 with PBS. Mix 

gently. (Sample dilution is based on the sample appearance or prior knowledge of 

sample. Dilute sample so OD falls on standard curve.) 

4.2.2 Transfer 1 mL of dilution (Section 4.2.1) to tube or cuvette. Run 

duplicates.

4.2.3 Transfer 1 mL PBS to tube or cuvette for instrument blank. 

4.2.4 Add 4 mL biuret reagent to each tube and mix gently. Let stand at room 

temperature for 30 to 45 minutes to allow for color development. 

4.2.5 Read OD at 540 nm, using blank to set zero. Read and record OD.

5. Interpretation of the Test Results

5.1 Calculation 

Determine sample value (either read from curve and multiply x dilution or enter data into 

computer program). Average test results of duplicate tests. Test results are acceptable if 

the duplicate test results vary no more than 5% from the mean and the protein value for 

the bovine serum control falls within 5% of the established value. 

5.2 Retest

If the OD of the diluted sample reads outside the end points on the standard curve, 

redilute sample and rerun the test.

6. Report of Test Results

Test results are reported following the current standard operating procedures. 

7. References

7.1 Robinson, H. W. and Hogden, C. G. (1940) J. Biol. Chem., vol. 135, pp. 707-725. 

7.2 Gornall, A. G., Bardawill, C. J., and David, M. M. (1949) J. Biol. Chem., vol. 

177, pp. 751-766. 

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Supplemental Assay Method for the Manual Determination of Protein Content

in Veterinary Biologics (Biuret)

7.3 Kibrick, A.C. (1958) Clin. Chem., vol. 4, pp. 232-236. 

7.4 Kingsley, G. R. (1939) J. Biol. Chem., vol. 131, pp. 197-200. 

8. Summary of Revisions 

Version .07 

• Updated coversheet and contact information. 

Version .06 

• Updated Contact information. 

• Replaced “reduced oxygen” method with “reverse osmosis” for Water. 

Version .05

• The Contact information has been updated. 

• References to specific products, such as media lot numbers, have been changed to 

optional. 

• All instructions for preparation of reagents and standards have been moved from 

appendices and Section 4.1 to Section 3.3. 

• The Quick Reference section has been removed

Version .04

• The document number has been changed from TCSAM0504 to SAM 504. 

Version .03 

This document was revised to clarify the practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the name of the contact person has changed.

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